In recent years, advances in biotechnology have made possible the production of desirable polypeptide products by inserting an appropriate heterologous gene into a host organism and subsequently culturing the organism to produce the polypeptide. In broad terms, these techniques involve the insertion of a structural gene coding for the desired polypeptide into a vector capable of stable existence in the cells of a host organism. The gene is inserted into the vector in a position relative to appropriate expression control sequences such that once within the host organism, the vector expresses the inserted gene to produce the polypeptide. Such vectors are referred to in the art as "expression vectors" and have been the subject of considerable research. The main thrust of the research has been to develop expression vectors which are compatible with prokaryotic host organisms such as bacteria (for example, Escherichia coli (E. coli)), and eukaryotic host organisms such as yeasts (for example, Saccharomyces cerevisiae) and cells of higher organisms (for example mammalian cells in tissue culture). A wide variety of polypeptides have been produced, such as animal and human hormones, enzymes and other useful proteins. The research has involved detailed studies of expression control sequences affecting expression, and in particular, promoter sequences, which are responsible for directing transcription of genetic material.
The commercial use of expression systems is hampered by the lethal or debilitating effect of toxic expression products upon the host organism. It has been recognised therefore that it is desirable to regulate heterologous gene expression. In a regulated expression system, the host organism can be cultured to produce a high cell density whilst expression of a gene in an inserted vector is kept at a low level. When the host organism reaches an appropriate cell density, expression may be induced, for example, by adding a material having a regulating effect on the culture medium. A large number of expression control sequences (including promoters) which allow a degree of expression regulation have been identified both for prokaryotic and eukaryotic host organisms. In published European Patent Application EP-A2-0073635, a yeast expression vector is described which makes use of the control sequences of the yeast phosphoglycerate kinase (PGK) gene and which allows expression level control by adjusting the level of fermentable carbon in the culture medium. Published European patent application EP-A2-0118393 describes a prokaryotic and eukryotic expression system based upon the control sequences of heat-shock genes derived from Drosophlia melanogaster. The control sequences include temperature-dependent promoters which allow for expression level control by adjusting the temperature of the culture medium. British patent specification No. 1557774 and published International patent application No. WO 84/01171 describe prokaryotic expression level control systems in which the average number of plasmids in each cell (the copy number) is controlled. Copy number control allows a regulation of the net gene expression occurring in each cell of the host organism.
The known regulated expression systems for eukaryotic host organisms do not have the ability to control expression over a wide range of expression levels. In many cases, it is not possible to reduce the concentration of toxic products, by promoter control alone, to a level at which the cell growth is unaffected and yet to allow for a significant production of the desired polypeptide when required.
The object of the present invention is to provide a eukaryotic expression vector capable of controlling the expression level of a heterologous gene, inserted in a host organism, over a wide range of expression levels.
The molecular biology of common brewers yeast, Saccharomyces cerevisiae, has been a target of considerable research effort. In particular the elucidation of the nature and operation of the mating type locus and its operation have been studied in depth. (See for reviews: Nasmyth Ann. Rev. Genet. (1982) 16 439-500 and Klar et al "Microbial Development" (1984) pub. Cold Spring Harbor Laboratory 151-195).
The mating type locus, which is located on chromosome III of the yeast genome, orchestrates the production of gene products necessary for the complex process of yeast mating. There are essentially three phenotypes of naturally occurring yeasts, a type haploids, .alpha. type haploids and a .alpha. diploids. A haploid can mate with a haploid of complementary phenotype to produce an a/.alpha. diploid. a/.alpha. diploids do not mate but are capable of sporulation under conditions of nutrient starvation. The mating type of the yeast is determined by a number of expression products of the yeast genome. The products expressed are controlled, in turn, by the expression products of the mating type locus. There are two active alleles of the yeast mating type locus, known as MATa and MAT.alpha.. Mating type locus allele MATa transcribes the gene known as al and mating type-locus allele MAT.alpha. transcribes genes known as .alpha.1 and .alpha.2. It is the expression of these genes which defines mating type. The a mating type is found where no MAT genes are expressed, the .alpha. type requires expression of both .alpha.1 and .alpha.2, and the a/.alpha. diploid requires expression of a1 and .alpha.2. The combination of gene expression products from the mating type locus acts on the yeast genome to promote or repress the production of specific gene products for mating.
We have discovered DNA sequences within the yeast genome which act as expression repressor operators when in the presence of mating type locus gene products.
According to the present invention there is provided a eukaryotic expression vector comprising an expression control sequence and a heterologous structural gene located relative to the expression control sequence such that the expression control sequence is capable of directing expression of the heterologous structural gene, characterised in that the expression control sequence includes a controllable repressor operator sequence comprising a DNA sequence which is capable of repressing expression in the presence of a gene product or a combination of gene products of the yeast mating type loci.
The vector of the invention allows control of the expression level of the heterologous structural gene over a wide range, thereby allowing growth of a culture whilst expression of the heterologous structural gene is substantially repressed prior to removing or reducing the level of the yeast mating type locus product or products to allow expression of the heterologous structural gene.
The eukaryotic expression vector of the invention may be an expression vector suitable for use in yeast or, for example, an expression vector suitable for use in a mammalian cell system. A suitable yeast expression vector may, for example, comprise a yeast expression vector such as the PGK promoter-based expression vectors described in European patent application EP-A2-0073635. Alternatively, the expression vector may be a vector suitable for the expression of gene products in a mammalian cell system such as an SV40 or bovine papilloma virus (BPV) expression vector.
The term "expression control sequence" as used herein denotes a sequence of DNA containing the control signals necessary to direct expression of the heterologous structural gene. The expression control sequence includes a promoter, and may include for example one or more upstream activator sequences (UAS) and other functional sequences.
The expression control sequence of the eukaryotic expression vector may comprise any functional eukaryotic promoter. The eukaryotic promoter may comprise a yeast promoter, in particular a yeast promoter which is not normally under mating type control. For instance, the yeast promoter may be a promoter derived from the yeast TRP1 ADH1, URA3.sup.+, HIS3.sup.+, CYC1 or PGK genes.
The controllable repressor operator sequence is inserted into the expression control sequence in a position where expression of the structural gene may be repressed. More than one copy of the controllable repressor operator sequence may be inserted into the expression control sequence to enhance repression. Preferably, the controllable repressor operator sequence is inserted between an upstream activator sequence (if present) and the "TATA box" of the yeast promoter. The controllable repressor operator sequence may lie upstream of an upstream activator sequence (if present).
The promoter may comprise a promoter suitable for use in an animal cell culture expression system. For example the promoter may comprise a viral promoter such as an SV40 promoter or a mammalian promoter such as a mammalian metallothionein promoter (for example, a mouse metallothionein promoter).
As used herein the term "heterologous structural gene" refers to a gene not naturally found in the host organism in which the expression vector is to be expressed. As used herein the term "polypeptide" denotes any polypeptide and includes hormones (such as growth hormones) and enzymes (such as chymosin).
The term "controllable repressor operator sequence" as used herein denotes an operator comprising a sequence of DNA capable, when inserted into the expression control sequence of a eukaryotic expression vector, of repressing expression in the presence of one or more of the gene products of the yeast mating type locus.
The gene products of the yeast mating type loci may be produced from more than one site in the yeast genome. For example copies of the relevant genes occur at silent mating type loci HML.alpha. and HMRa. Gene products from these loci may be used to effect repression by the controllable repressor operator sequence.
The controllable repressor operator sequence may comprise a DNA sequence which represses expression in the presence of a combination of the a1 and .alpha.2 gene products of the MAT.alpha. and MATa yeast mating type locus alleles. The DNA sequence has been shown to occur repeatedly in those genes specific to haploid yeast types. In a diploid yeast, the a1 and .alpha.2 gene products repress expression of haploid specific genes.
In broad terms, a DNA sequence comprises a double-stranded sequence of about twenty base pairs having subsequences of about seven base pairs at opposite ends and in complementary strands of the sequence, wherein the subsequences are substantially inverted repeats each of the other. Preferably the DNA is selected from one of the following sequences
______________________________________ CAATGTAGAAAAGTACATCA (MAT .alpha.1) GCTTGTTAATTTACACATCA (STE5(-196)) TCATGTACTTTTCTGCATCA (STE5(-179)) CCGCGTTAAAACCTACATCA (HO -1752) TTATGTTAAAAGTTACATCC (HO -1391) GCCTGCGATGAGATACATCA (HO -1328) TAGAGTGAAAAAGCACATCG (HO -1208) TCATGTATTCATTCACATCA (HO -736) ACATGTCTTCAACTGCATCA (HO -669) TCGTGTATTTACTTACATCA (HO -576) TCATGTTATTATTTACATCA (HO -411) TCATGTCCACATTAACATCA (HO -371) and GCGTTTAGAACGCTTCATCA (HO -150) ______________________________________
wherein (using the standard notation for nucleotide bases employed throughout this specification) A denotes adenine, T denotes thymine, G denotes guanine and C denotes cytosine. (The source of the sequence and the position of the sequence within the gene are shown in parenthesis. It will be understood that the sequences shown above represent a single strand of a double stranded portion of DNA, the strand not shown being complementary to the shown strand.).
The prevalence of the DNA sequence in haploid specific genes allows for the establishment of a statistical consensus sequence which provides a statistical best fit of the individual sequences. The controllable repressor operator sequence may comprise a DNA sequence having substantially the following nucleotide base sequence: EQU TC(A or G)TGTNN(A or T)NANNTACATCA
wherein N denotes a nucleotide base selected from adenine, thymine, guanine and cytosine.
Alternatively, the controllable repressor operator sequence may comprise a DNA sequence which represses expression in the presence of the .alpha.2 gene product of the MAT.alpha. yeast mating type locus allele. The DNA sequence has been shown to occur repeatedly in those genes specific to a haploid yeast types. In .alpha. yeast cells, genes essential for a mating are repressed by the .alpha.2 gene product.
In broad terms, such a DNA sequence comprises a double-stranded sequence of about thirty-three base pairs having subsequences of about ten base pairs at opposite ends and in complementary strands of the sequence, wherein the subsequences are substantially inverted repeats each of the other. Preferably the DNA sequence is selected from one of the following sequences:
__________________________________________________________________________ GTGTGTAATTACCCAAAAAGGAAATTTACATGT (MFA1) GCATGTAATTACCGTAAAAGGAAAT-TACATGG (BAR1) and TCATGTACTTACCCAATTAGGAAATTTACATGG (STE2) __________________________________________________________________________
(The source of the gene is shown in parenthesis. It will be understood that the sequences shown above represent a single strand of a double stranded portion of DNA, the strand not shown being complementary to the shown strand.).
The DNA sequence of the .alpha.2 product-controllable repressor operator sequence occurs more than once in the yeast genome and, again, it is possible to assign a consensus sequence. The controllable repressor operator sequence may comprise a DNA sequence having substantially the following nucleotide base sequence: EQU GCATGTAATTACCCAAAAAGGAAATTTACATGG
The DNA sequence of the controllable repressor operator sequence may comprise the whole or an operative part of any of the sequences mentioned above, or a functional equivalent thereof.
The sequence may be obtained from natural or mutant yeast genes which are under mating type control, for instance by appropriate restriction enzyme digestion. Preferably, however, the sequence is prepared by chemical synthesis and, where appropriate, ligation of two or more synthetic oligonucleotides.
In a second aspect of the invention we provide a controllable repressor operator sequence capable, when inserted into the expression control sequence of a eukaryotic expression vector, of repressing expression in the presence of a combination of the a1 and .alpha.2 gene products of the MATa and MAT.alpha. yeast mating type allele.
In a third aspect of the invention we provide a controllable repressor operator sequence capable, when inserted into the expression control sequence of a eukaryotic expression vector, of repressing expression in the presence of the .alpha.2 gene product of the MAT.alpha. yeast mating type locus.
The sequences of the second and third aspects of the invention may each be provided with a linker at each end to facilitate the insertion of the sequence into a suitable site in the expression control sequence of a eukaryotic expression vector.
The vectors of the first aspect of the invention may be used to transform or transfect eukaryotic host organisms, for example, by methods well known in the art. The eukaryotic host organism may be a yeast, such as Saccharomyces cerevisiae or a higher eukaryotic host organism such as a culture of animal cells.
In a fourth aspect of the invention we provide a eukaryotic host organism transformed with an expression vector according to the first aspect of the invention.
Preferably the host organism is transformed with a eukaryotic expression vector according to the first aspect of the invention and further comprises means for providing a controllable supply of either a combination of the .alpha.2 and a1 protein or the .alpha.2 protein alone to cause repression of transcription.
The host organism may be transformed with a eukaryotic expression vector according to the present invention and a second vector, allowing the controllable production of either a combination of the a1 and .alpha.2 gene products or the .alpha.2 gene product alone. The second vector may, for example, comprise a vector capable of producing a controllable level of either a combination of the a1 and .alpha.2 gene products or of the .alpha.2 gene product alone. A suitable such vector is a temperature-dependent mutant including a MAT gene or, for example, a temperature-sensitive mutant of a SIR gene. A SIR gene regulates mating type gene expression from silent mating type loci HML.alpha. and HMRa. These may be used to provide a regulated supply of the controlling protein or proteins.
Alternatively the yeast strain used as host for the eukaryotic expression vector of the invention may, for example, carry a temperature sensitive mutation in the a1 and/or .alpha.2 repressor gene. Thus if transformed cells are grown at a permissive temperature the a1 and/or .alpha.2 proteins are functional and repress transcription of the heterologous structural gene, whereas at a restrictive temperature the repressor proteins are inactive, transcription of the heterologous structural gene is not repressed and the heterologous gene product is expressed.
The vector of the present invention and/or the said second vector may be present within the transformed eukaryotic host cells in an episomal form or may be incorporated into the chromosome of the host organism.
Alternatively, the combination of the a1 and .alpha.2 gene products or the .alpha.2 gene product alone may be introduced into the culture medium from an external source or may be produced in controllable manner on a suitable modified expression vector of the present invention.
In a fifth aspect of the invention we provide a method for preparing a polypeptide comprising culturing a eukaryotic host organism transformed with a vector according to the invention in the presence of one or more gene products of the yeast mating type locus capable of repressing expression of the heterologous structural gene, until a predetermined cell density has been established, and subsequently reducing the level of the gene product or products of the yeast mating locus, thereby allowing expression of the heterologous structural gene and production of the polypeptide.
In a sixth aspect of the invention we provide a eukaryotic expression vector of the present invention in which a restriction site suitable for the insertion of a heterologous structural gene exists in place of the heterologous structural gene. Preferably the restriction site is unique in the vector. A gene coding for a desired polypeptide may readily be ligated into such an expression vector to produce a vector according to the first aspect of the invention.